The invention relates to the area of regulation of human S-acyl fatty acid synthase thioesterase-like enzyme to provide therapeutic effects.
The substrate specificity of fatty acid synthase can be modulated through the modification of fatty acid synthase by the enzyme S-acyl fatty acid synthase thioesterase. This modification causes fatty acid synthesis to be shifted from long chain fatty acids, which typically have 16, 18 or more carbon atoms in the fatty acid carbon chain, to medium chain fatty acids, typically having between 8-14 carbon atoms in the fatty acid chain. See U.S. Pat. No. 5,147,792. For example, rat medium-chain specific S-acyl fatty acid synthase thioesterase modulates the substrate specificity of fatty acid synthase through the preferential fatty acid chain termination, via premature release from the fatty acid synthase multifunctional complex (1) of a growing acyl chain. This reaction also has been observed in a transgenic plant (2).
In humans, the complex regulation of lipid metabolism involves various fatty acids and dietary triglycerides. Because medium chain fatty acids contribute to raising low density lipoprotein (LDL) blood cholesterol levels (3, 4, 5) in humans, they are generally undesirable. High levels of LDL blood cholesterol in turn have been implicated as associated with a number of conditions and diseases, including cardiovascular disease, hyperlipidemia, obesity, and diabetes. Regulation of S-fatty acid synthase thioesterase to lower LDL levels therefore has important implications for treatment of these.
It is an object of the invention to provide reagents and methods of regulating a human S-acyl fatty acid synthase thioesterase-like enzyme. This and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention is a cDNA encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16.
Another embodiment of the invention is an expression vector comprising a polynucleotide which encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16.
Yet another embodiment of the invention is a host cell comprising an expression vector which encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16.
A further embodiment of the invention is a purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16.
Still another embodiment of the invention is a fusion protein comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16.
Even another embodiment of the invention is a method of producing a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16. A host cell comprising an expression vector which encodes the polypeptide is cultured under conditions whereby the polypeptide is expressed. The polypeptide is isolated.
Yet another embodiment of the invention is a method of detecting a coding sequence for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16. A polynucleotide comprising 11 contiguous nucleotides of SEQ ID NOS:12, 13, or 14 is hybridized to nucleic acid material of a biological sample, thereby forming a hybridization complex. The hybridization complex is detected.
Still another embodiment of the invention is a kit for detecting a coding sequence for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16. The kit comprises a polynucleotide comprising 11 contiguous nucleotides of SEQ ID NOS:12, 13, or 14 and instructions for detecting the coding sequence.
Even another embodiment of the invention is a method of detecting a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16. A biological sample is contacted with a reagent that specifically binds to the polypeptide to form a reagent-polypeptide complex. The reagent-polypeptide complex is detected.
A further embodiment of the invention is a kit for detecting a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16. The kit comprises an antibody which specifically binds to the polypeptide and instructions for detecting the polypeptide.
Another embodiment of the invention is a method of screening for agents that can regulate the activity of an S-acyl fatty acid synthase thioesterase-like enzyme. A test compound is contacted with a polypeptide comprising an amino acid sequence selected from the group consisting of: (1) amino acid sequences which are at least about 50% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16 and (2) the amino acid sequences shown in SEQ ID NOS:2, 14, and 16. Binding of the test compound to the polypeptide is detected. A test compound that binds to the polypeptide is thereby identified as a potential agent for regulating activity of the S-acyl fatty acid synthase thioesterase-like enzyme.
Even another embodiment of the invention is a method of screening for agents which regulate an activity of a human the S-acyl fatty acid synthase thioesterase-like enzyme. A test compound is contacted with a polypeptide comprising an amino acid sequence selected from the group consisting of: (1) amino acid sequences which are at least about 50% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16 and (2) the amino acid sequences shown in SEQ ID NOS:2, 14, and 16. An activity of the polypeptide is detected. A test compound that increases the activity of the polypeptide is thereby identified as a potential agent for increasing the activity of the human S-acyl fatty acid synthase thioesterase-like enzyme. A test compound that decreases the activity of the polypeptide is thereby identified as a potential agent for decreasing the activity of the human S-acyl fatty acid synthase thioesterase-like enzyme.
A further embodiment of the invention is a method of screening for agents that regulate an activity of a human S-acyl fatty acid synthase thioesterase-like enzyme. A test compound is contacted with a product encoded by a polynucleotide which comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:12, 13, and 15. Binding of the test compound to the product is detected. A test compound that binds to the product is thereby identified as a potential agent for regulating the activity of the human S-acyl fatty acid synthase thioesterase-like enzyme.
Still another embodiment of the invention is a method of reducing activity of a human S-acyl fatty acid synthase thioesterase-like enzyme. A cell is contacted with a reagent which specifically binds to a product encoded by a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1. The activity of a human S-acyl fatty acid synthase thioesterase-like enzyme is thereby reduced.
Another embodiment of the invention is a pharmaceutical composition, comprising a reagent which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16 and a pharmaceutically acceptable carrier.
Still another embodiment of the invention is a pharmaceutical composition. A reagent that specifically binds to a product of a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS:12, 13, and 15 and a pharmaceutically acceptable carrier.
Even another embodiment of the invention is a pharmaceutical composition comprising an expression vector encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16 and a pharmaceutically acceptable carrier.
Yet another embodiment of the invention is a method of treating a disorder selected from the group consisting of cardiovascular disease, hyperlipidemia, obesity, and diabetes. A therapeutically effective dose of a reagent that inhibits a function of a human S-acyl fatty acid synthase thioesterase-like enzyme is administered to a patient in need thereof. Symptoms of the disorder are thereby ameliorated.
A further embodiment of the invention is an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide encoding a protein that comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16, (b) a polynucleotide comprising a nucleotide selected from the group consisting of SEQ ID NOs:12, 13, and 15, (c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) or (b); (d) a polynucleotide having a nucleic acid sequence that deviates from the nucleic acid sequences specified in (a)-(c) due to the degeneration of the genetic code, and (e) a polynucleotide that represents a fragment, derivative, or allelic variation of a nucleic acid sequence specified in (a)-(d).
Yet another embodiment of the invention is an expression vector comprising a polynucleotide selected from the group consisting of: (a) a polynucleotide encoding a protein that comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16, (b) a polynucleotide comprising a nucleotide selected from the group consisting of SEQ ID NOs:12, 13, and 15, (c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) or (b); (d) a polynucleotide having a nucleic acid sequence that deviates from the nucleic acid sequences specified in (a)-(c) due to the degeneration of the genetic code, and (e) a polynucleotide that represents a fragment, derivative, or allelic variation of a nucleic acid sequence specified in (a)-(d).
Even another embodiment of the invention is a host cell comprising an expression vector comprising a polynucleotide selected from the group consisting of: (a) a polynucleotide encoding a protein that comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16, (b) a polynucleotide comprising a nucleotide selected from the group consisting of SEQ ID NOs:12, 13, and 15, (c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) or (b); (d) a polynucleotide having a nucleic acid sequence that deviates from the nucleic acid sequences specified in (a)-(c) due to the degeneration of the genetic code, and (e) a polynucleotide that represents a fragment, derivative, or allelic variation of a nucleic acid sequence specified in (a)-(d).
Still another embodiment of the invention is a preparation of antibodies that specifically bind to a polypeptide selected from the group consisting of (a) an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16 and (b) biologically active variants thereof.
Yet another embodiment of the invention is an antisense oligonucleotide that hybridizes to a polynucleotide selected from the group consisting of (a) a polynucleotide encoding a protein that comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 14, and 16, (b) a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS:12, 13, and 15, (c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) or (b), (d) a polynucleotide having a nucleic acid sequence that deviates from the nucleic acid sequences specified in (a)-(c) due to the degeneration of the genetic code, and (e) a polynucleotide that represents a fragment, derivative, or allelic variation of a nucleic acid sequence specified in (a)-(d).
The invention thus provides a human S-acyl fatty acid synthase thioesterase-like enzyme which can be used to identify test compounds which may act, for example, as agonists or antagonists at the enzyme""s active site. Human S-acyl fatty acid synthase thioesterase-like enzyme and fragments thereof also are useful in raising specific antibodies which can block the enzyme and effectively reduce its activity.